Flowable acellular tissue matrix products and methods of production

ABSTRACT

Tissue product compositions and methods for treating a patient are provided. The tissue product composition may include a flowable carrier including a hyaluronic acid based material and acellular tissue matrix particles mixed within the carrier. Methods of producing the tissue product composition and an injection device filled with the tissue product composition are also provided.

The present disclosure claims priority under 35 USC § 119 to U.S.Provisional Application 62/574,678, which was filed on Oct. 19, 2017 andis herein incorporated by reference.

The present disclosure relates to tissue matrices, and moreparticularly, to injectable materials that may be used for surgical ormedical procedures including tissue regeneration for aesthetic ornon-aesthetic purposes.

There is currently a need for improved injectable materials for tissuetreatment. For example, to treat various facial features (e.g., lines,wrinkles, insufficient volume, or less than desirable shapes or forms),injectable materials such as hyaluronic-acid-based materials may beused. Such materials, however effective, may provide only temporaryimprovements due to eventual resorption by the body. Although work hasbeen done to develop hyaluronic acid (HA) materials that last longer invivo before resorption by, for example, cross-linking the HA, thecurrent materials will still inevitably be resorbed by the bodyfollowing injection.

There exists an unmet need for tissue product compositions that canproduce longer lasting effects while being suitable for injectionthrough a syringe or otherwise easily handled injection device orimplantation system.

Accordingly, the present disclosure provides tissue product compositionshaving acellular tissue matrix particles mixed within a flowable carriercomprising a hyaluronic acid based material. The disclosure alsoprovides injection devices with such compositions and methods forproducing such compositions. In addition, methods of treatment usingsuch compositions are provided.

The present disclosure provides tissue product compositions. The tissueproduct composition may include a flowable carrier comprising ahyaluronic acid based material and a plurality of acellular tissuematrix particles mixed with the carrier.

The present disclosure also provides injection devices. The injectiondevice may include a syringe including a reservoir defining a volume anda needle fluidly coupled to the reservoir and a tissue productcomposition held in the reservoir. The tissue product composition mayinclude a flowable carrier comprising a hyaluronic acid based materialand a plurality of acellular tissue matrix particles mixed within thecarrier.

The present disclosure also provides methods of producing a tissueproduct composition. The method may include mixing a plurality ofacellular tissue matrix particles within a flowable carrier comprising ahyaluronic acid based material.

The present disclosure also provides methods of treatment using thedisclosed tissue product compositions.

It may be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate exemplary embodiments of thepresent disclosure and together with the description, serve to explainthe principles of the disclosure.

FIG. 1 is a flow chart illustrating an exemplary embodiment of a methodfor producing tissue products provided in accordance with the presentinvention;

FIG. 2 is a graph illustrating exemplary embodiments of tissue productcompositions having different particle size distributions;

FIG. 3 is a graph illustrating an amplitude sweep report for a tissueproduct composition comprising acellular tissue matrix particles thatare not mixed within a flowable carrier;

FIG. 4 is a graph illustrating G′ and G″ values at 5 Hz for varioustissue product compositions;

FIG. 5 is a graph illustrating compressive extension and load behaviorfor known tissue product compositions compared to an exemplaryembodiment of a tissue product composition formed in accordance with thepresent invention;

FIG. 6 is a graph illustrating injection forces using different needlesizes for various tissue product compositions;

FIG. 7 is a microscope photograph of trichrome stained collagen of anexemplary embodiment of a tissue product composition formed inaccordance with the present invention;

FIG. 8 is a microscope photograph of a tissue product compositioncomprising acellular tissue matrix particles that are not mixed within aflowable carrier;

FIG. 9 is a microscope photograph of an exemplary embodiment of a tissueproduct composition comprising acellular tissue matrix particles thatare mixed with a flowable carrier in accordance with the presentinvention;

FIG. 10 is a microscope photograph of another exemplary embodiment of atissue product composition formed in accordance with the presentinvention;

FIG. 11 is a microscope photograph of yet another tissue productcomposition formed in accordance with the present invention;

FIG. 12 is a microscope photograph of yet another tissue productcomposition formed in accordance with the present invention;

FIG. 13 is a microscope photograph of yet another tissue productcomposition formed in accordance with the present invention;

FIG. 14 is a microscope photograph of yet another tissue productcomposition formed in accordance with the present invention;

FIG. 15 is a microscope photograph of yet another tissue productcomposition formed in accordance with the present invention;

FIG. 16 is a microscope photograph illustrating the integration of atissue product composition into host dermal tissue four weeks postimplantation;

FIGS. 17A-D is a group of microscope photographs comparing biologicalresponses observed for implanted tissue product compositions with andwithout a flowable hyaluronic acid carrier;

FIGS. 18A and 18B are microscope photographs comparing dermal thicknesspost-implantation over time;

FIG. 19 is a graph illustrating volume retention of implanted tissueproduct compositions;

FIG. 20 is a graph illustrating the collagenase resistance ofcross-linked tissue product compositions; and

FIG. 21 is a graph illustrating the volume retention of implantedcross-linked tissue product compositions cross-linked using differentconcentrations of EDC.

FIG. 22 is a graph illustrating the biological responses in implantedcross-linked tissue product compositions cross-linked using differentconcentrations of EDC.

DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain exemplary embodimentsaccording to the present disclosure, certain examples of which areillustrated in the accompanying drawings. Wherever possible, the samereference numbers will be used throughout the drawings to refer to thesame or like parts.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Similarly, the use of the term “comprising,” as well asother forms, such as “comprises,” is also not limiting. Any rangedescribed herein will be understood to include the endpoints and allvalues between the endpoints.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose.

As used herein, the term “acellular tissue matrix” refers to anextracellular matrix derived from human or animal tissue, wherein thematrix retains a substantial amount of natural collagen andglycoproteins needed to serve as a scaffold to support tissueregeneration. “Acellular tissue matrices” are different from purifiedcollagen materials, such as acid-extracted purified collagen, which aresubstantially void of other matrix proteins and do not retain thenatural micro-structural features of tissue matrix due to thepurification processes. Although referred to as “acellular tissuematrices,” it will be appreciated that such tissue matrices may combinewith exogenous cells, including, for example, stem cells, or cells froma patient in whom the “acellular tissue matrices” may be implanted.Further, it should be appreciated that “acellular tissue matrixparticles” refers to particulate of an acellular tissue matrix, as willbe described further herein.

“Acellular” or “decellularized” tissue matrices will be understood torefer to tissue matrices in which no cells are visible using lightmicroscopy.

Various human and animal tissues may be used to produce products fortreating patients. For example, various tissue products forregeneration, repair, augmentation, reinforcement, and/or treatment ofhuman tissues that have been damaged or lost due to various diseasesand/or structural damage (e.g., from trauma, surgery, atrophy, and/orlong-term wear and degeneration) have been produced. Such products mayinclude, for example, acellular tissue matrices, tissue allografts orxenografts, and/or reconstituted tissues (i.e., at least partiallydecellularized tissues that have been seeded with cells to produceviable materials).

The tissue products may include acellular tissue matrix particles fromdermal or other tissues that have been processed to remove at least someof the cellular components. In some cases, all, or substantially all,cellular materials are removed, thereby leaving correspondingextracellular matrix proteins. While dermal tissue is primarilydescribed herein as being the source tissue for exemplary embodiments ofacellular tissue matrix particles, it should be appreciated that theacellular tissue matrix particles described herein can originate fromother tissue types. Other exemplary tissue types include, but are notlimited to: adipose tissue, small intestine submucosa (SIS) tissue,muscle tissue, vascular tissue, and bone tissue.

The source tissues described herein may be derived from human or animalsources. For example, tissue may be obtained from cadavers. In addition,human tissue could be obtained from live donors (e.g., autologoustissue). Tissue may also be obtained from animals such as pigs, monkeys,or other sources. If animal sources are used, the tissues may be furthertreated to remove antigenic components such as 1,3-alpha-galactosemoieties, which are present in pigs and other mammals, but not humans orprimates. In addition, the tissue may be obtained from animals that havebeen genetically modified to remove antigenic moieties. See Xu, Hui. etal., “A Porcine-Derived Acellular Dermal Scaffold that Supports SoftTissue Regeneration: Removal of Terminal Galactose-α-(1,3)-Galactose andRetention of Matrix Structure,” Tissue Engineering, Vol. 15, 1-13(2009), which is hereby incorporated by reference in its entirety.

As used herein, a “hyaluronic acid based material” is a materialcomprising hyaluronic acid (HA). HA refers to hyaluronic acid and canalso refer to any salts thereof, including, but not limited to, sodiumhyaluronate, potassium hyaluronate, magnesium hyaluronate, calciumhyaluronate, and combinations thereof. Both HA and pharmaceuticallyacceptable salts thereof can be included in the hyaluronic acid basedmaterial. Exemplary HA based materials are commercially sold asJUVEDERM® and JUVEDERM VOLUMA®. It should be appreciated that thehyaluronic acid based material may include additional agents such as,for example, lidocaine.

All numbers herein expressing “molecular weight” of HA are to beunderstood as indicating the weight average molecular weight (Mw) inDaltons.

The molecular weight of HA is calculated from an intrinsic viscositymeasurement using the following Mark Houwink relation: IntrinsicViscosity (m3/kg)=9.78×10⁻⁵×Mw^(0.690). The intrinsic viscosity ismeasured according to the procedure defined European Pharmacopoeia (HAmonograph No 1472, 01/2009).

High molecular weight HA as used herein describes a HA material having amolecular weight of at least about 1.0 million Daltons (mw 10⁶ Da or 1MDa) to about 4.0 MDa. High molecular weight HA that may be incorporatedin the present tissue product compositions may have a molecular weightin the range about 1.5 MDa to about 3.0 MDa, or the high molecularweight HA may have a weight average molecular weight of about 2.0 MDa.In another example, the high molecular weight HA may have a molecularweight of about 3.0 MDa.

Low molecular weight HA as used herein describes a HA material having amolecular weight of less than about 1.0 MDa. Low molecular weight HA canhave a molecular weight of between about 200,000 Da (0.2 MDa) to lessthan 1.0 MDa, for example, between about 300,000 Da (0.3 M Da) to about750,000 Da. (0.75 MDa), up to but not exceeding 0.99 MDa. Preferably,there is no overlap between the molecular weight distribution of the lowand high molecular weight HA materials. Preferably, the mixture of thelow molecular weight HA and high molecular weight HA has a bimodalmolecular weight distribution. The mixture may also have a multi-modaldistribution.

In one aspect of the invention, the tissue product compositions compriseHA having a high molecular weight component and a low molecular weightcomponent, and the high molecular weight component may have a weightaverage molecular weight at least twice the weight average molecularweight of the low molecular weight component. For example, the molecularweight ratio of the high molecular weight HA to the low molecular weightHA in the composition may be at least 2:1. For example, a tissue productcomposition may include an HA having a low molecular weight componenthaving a weight average molecular weight of about 500,000 Da, and a highmolecular weight component having a weight average molecular weight ofabout, or at least about, 1.0 MDa. In another example, a tissue productcomposition in accordance with the invention may include an HA having alow molecular weight component having a weight average molecular weightof about 800,000 Da, and a high molecular weight component having aweight average molecular weight of about, or at least about, 1.6 MDa. Itshould be appreciated that many different types of HA may beincorporated in the tissue product composition, and the foregoingexamples are not intended to be limiting.

In some exemplary embodiments, the HA may be cross-linked using one ormore suitable crosslinking agents. The crosslinking agent may be anyagent known to be suitable for crosslinking polysaccharides and theirderivatives via their hydroxyl groups. Suitable crosslinking agentsinclude but are not limited to, 1,4-butanediol diglycidyl ether (or1,4-bis(2,3-epoxypropoxy)butane or 1,4-bisglycidyloxybutane, all ofwhich are commonly known as BDDE), 1,2-bis(2,3-epoxypropoxy)ethylene,1-(2,3-epoxypropyI)-2,3-epoxycyclohexane, and1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (commonlyknown as EDC). Other suitable hyaluronan crosslinking agents includemultifunctional PEG-based crosslinking agents like pentaerythritoltetraglycidyl ether (PETGE), divinyl sulfone (DVS),1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO),(phenylenebis-(ethyl)-carbodiimide and 1,6 hexamethylenebis(ethylcarbodiimide), adipic dihydrazide (ADH),bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA),1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, or combinations thereof.

In one exemplary embodiment of a tissue product composition formed inaccordance with the present invention, the tissue product compositionincludes a flowable carrier comprising a hyaluronic acid based materialand a plurality of acellular tissue matrix particles mixed within thecarrier. In some exemplary embodiments, the flowable carrier comprisesHA that has not been mixed with additional agents; in other exemplaryembodiments, the flowable carrier comprises HA mixed with additionalagents. Additional agents may include, but are not limited to,anesthetic agents for example, aminoamide local anesthetic and saltsthereof or an aminoester local anesthetic and salts thereof. Forexample, procaine, chloroprocaine, cocaine, cyclomethycaine,cimethocaine, propoxycaine, procaine, proparacaine, tetracaine, or saltsthereof, or any combination thereof. In some embodiments, anestheticagents may comprise articaine, bupivacaine, cinchocaine, etidocaine,levobupivacaine, lidocaine, mepivacaine, piperocaine, prilocaine,ropivacaine, trimecaine, or salts thereof, or any combination thereof.

The flowable carrier may initially be in the form of a flowable liquidsolution that can be mixed with the tissue matrix particles to form aslurry. The formed slurry can then be loaded into a syringe or otherinjection device for administration to a patient. In some exemplaryembodiments, the flowable carrier may be a non-crosslinked HA in anamount sufficient to provide improved injectability of the tissueproduct composition. While the flowable carrier is described herein ascomprising HA, it is contemplated that other glycosaminoglycans (GAGs)may be utilized as the flowable carrier, such as HSGAG, CSGAG, and/orkeratin sulfate type GAGs.

The acellular tissue matrix particles may originate from a human oranimal tissue matrix, as previously described. Suitable tissue sourcesmay include allograft, autograft, or xenograft tissues. When xenograftsare used, the tissue may include tissues from animals including porcine,cow, dog, cat, domestic or wild sources, and/or any other suitablemammalian or non-mammalian tissue source. In some exemplary embodiments,the acellular tissue matrix particles may originate from a source dermalmatrix taken from an animal, such as a pig. In one exemplary embodiment,the source dermal matrix may comprise one or more layers of skin thathave been removed from an organism. The size and shape of the sourcetissue matrix may be varied, according to known methods, to producediffering amounts of acellular tissue matrix particles, as will bedescribed further herein.

The source tissue may be harvested from animal sources using anydesirable technique, but may be generally obtained using, if possible,aseptic or sterile techniques. The tissue may be stored in cold orfrozen conditions or may be immediately processed to prevent anyundesirable changes due to prolonged storage.

Acellular tissue matrices can provide a suitable tissue scaffold toallow cell ingrowth and tissue regeneration. In the context of skinlines, wrinkles, etc., a tissue scaffold could represent a long-termsolution to filling in lost volume without needing to be replaced likeHA or other temporary filler materials. The tissue scaffolds can beinjected into existing or damaged skin to allow production of a thickerdermis at the injection site. Tissue scaffolds may also be useful inother medical applications, such as large volume tissue repair.

One particular problem that has been identified with using an acellulartissue matrix is the difficulty of placement, such as injecting,acellular tissue matrix material in its natural form, due to the networkformed by the tissue matrix. While surgical implantation is a suitableoption for implanting acellular tissue matrix materials to repaircertain areas of the body, injection may be preferred for someapplications. Particulating the acellular tissue matrix was found to bean improvement for application and injection, compared to applying theacellular tissue matrix in its natural form, but it was found that evenparticulated pure acellular tissue matrix was not easily applied orinjected into a patient. Particularly, application of the particulatedtissue matrix material was found to be difficult to control, due to thetendency of the particulated tissue matrix material to spread. Further,the injection force required to inject particulated acellular tissuematrix was found to be relatively high, and it was found to berelatively difficult to inject all of the particulated tissue matrixloaded into an injection device, such as a syringe.

To address some of the previously described problems of injectingacellular tissue matrix materials, exemplary embodiments describedherein provide tissue product compositions including acellular tissuematrix particles mixed within a flowable carrier comprising a hyaluronicacid based material. The formed tissue product composition can be moreeasily applied than pure acellular tissue matrix particles, as will bedescribed further herein, while maintaining characteristics thatencourage tissue growth in the implantation and/or injection area.

The tissue product compositions described herein may be used to treat avariety of different anatomic sites. In one exemplary embodiment, thetissue product compositions may be formed as an injectable tissueproduct composition suitable for small-volume implantations, e.g., totreat lines, wrinkles, voids, or divots, to add volume (e.g., byincreasing dermal thickness), or replace small volumes of lost tissue.In other exemplary embodiments, the tissue product compositions may beformed as a putty or paste suitable for larger volume implantations suchas repairing large areas of structural tissue, breast tissuereplacement, or any other area where a relatively large volume ofdamaged and/or lost tissue must be repaired and/or replaced. In otherexemplary embodiments, the tissue product compositions may be formed asan injectable tissue product composition suitable for use inapplications where HA or other fillers would be utilized, with theinjected tissue product composition representing a long-term, ratherthan short-term, treatment. In yet other exemplary embodiments, thetissue product compositions may be originated from more than onesources. For example, a tissue product composition can be originatedfrom a dermal matrix and an adipose matrix.

Referring now to FIG. 1, an exemplary embodiment of a method 100 forproducing a tissue product composition in accordance with the presentinvention is illustrated. The method 100 generally includes mixing aplurality of acellular tissue matrix particles within a flowable carriercomprising a hyaluronic acid based material to produce the tissueproduct composition. In some exemplary embodiments, the method 100 mayalso include processing 102 a source tissue matrix, such as a sourcedermal matrix, to produce the acellular tissue matrix particles prior tomixing 108 the acellular tissue matrix particles with the flowablecarrier. In one exemplary embodiment, the method 100 includes initiallydecontaminating 101 the source tissue matrix to remove contaminants,such as dirt or other debris or microbes, to clean and prepare thesource tissue matrix for processing 102. In some exemplary embodiments,the decontaminating 101 may comprise washing the source tissue matrix.For example, the source tissue matrix may be washed with one or morerinses with various biocompatible buffers. For example, suitable washsolutions may include saline, phosphate buffered saline, or othersuitable biocompatible materials or physiological solutions. In oneexemplary embodiment, water may be used as a rinsing agent to furtherbreak the cells, after which phosphate buffered saline, or any othersuitable saline solution, may be introduced to allow the matrix proteinsto return to biocompatible buffers. A large variety of other suitabledecontamination processes are known, so further description of thedecontamination is omitted for brevity.

The source tissue matrix is processed 102 to break up the source tissuematrix into a plurality of tissue matrix particles. The source tissuematrix may, generally speaking, be in a form that is initiallyunsuitable for easy application or injection through a syringe or otherinjection device, due to the structure of the source tissue matrix. Forexample, a source dermal matrix may be in the form of a planar sheet,which is not easily worked with or loaded into or expelled from aninjection device. To process 102 the source tissue matrix, a grinder orother mechanical separation device may be employed to break up thesource tissue matrix into tissue matrix particles by grinding, blending,chopping, grating, and/or other mechanical agitation of the sourcetissue matrix. In some exemplary embodiments, the processing 102 mayproduce tissue matrix particles having many different particle sizes(diameters), which will be described further herein.

In some cases, the processing 102 may be performed by mechanicallyprocessing the tissue with the addition of little or no washing orlubricating fluids. For example, the tissue may be mechanicallyprocessed by grinding or blending without the use of solvents. Whengrinding the tissue requires moisture, for example, water may be usedover other solutions, such as saline or phosphate buffered saline.Alternatively, the tissue may be processed by adding a certain quantityof solvent that is biocompatible, such as saline (e.g., normal saline,phosphate buffered saline, or solutions including salts and/ordetergents). Other solutions that facilitate cell lysis may also beappropriate.

The method 100 may further include de-cellularizing 103 the sourcetissue matrix or produced tissue matrix particles to removesubstantially all of the native cellular material, which may cause anantigenic response in the patient following injection. Thede-cellularization 103 may include a number of suitable processes. Forexample, suitable methods for removing cells from the source tissuematrix or tissue matrix particles may include treatment with detergentssuch as deoxycholic acid, polyethylene glycols, or other detergents atconcentrations and times sufficient to disrupt cells and/or removecellular components. In some exemplary embodiments, the thede-cellularization 103 may occur after processing 102 the source tissuematrix into tissue matrix particles; in other exemplary embodiments, thede-cellularization 103 may occur prior to processing 102 the sourcetissue matrix into tissue matrix particles. In still other exemplaryembodiments, de-cellularization 103 may take place both before and afterprocessing 102 the source tissue matrix into tissue matrix particles.Regardless of when de-cellularization 103 takes place, acellular tissuematrix particles should be produced following both the processing 102and de-cellularization 103.

Optionally, the method 100 may include additionally decontaminating 104the formed acellular tissue matrix particles to remove contaminants thatmay have been inadvertently introduced or produced during the processing102 and/or de-cellularization 103. The decontamination 104 may besimilar to the previously described decontamination 101, or may compriseadditional washing or treatment of the acellular tissue matrixparticles. For example, additional washing or treatment may be performedto remove antigenic materials such as alpha-1,3-galactose moieties,which may be present on non-primate animal tissues. In addition, during,before, and/or after the washing steps, additional solutions or reagentsmay be used to process the material. For example, enzymes, detergents,and/or other agents may be used in one or more steps to further removecellular materials or lipids, remove antigenic materials, and/or reducethe bacteria or other bioburden of the material. For example, one ormore washing steps may be included using detergents, such as sodiumdodecylsulfate or TRIS to assist in cell and lipid removal. In addition,enzymes such as lipases, DNAses, RNAses, alpha-galactosidase, or otherenzymes may be used to ensure destruction of nuclear materials, antigensfrom xenogenic sources, residual cellular components and/or viruses.Further, acidic solutions and/or peroxides may be used to help furtherremove cellular materials and destroy bacteria and/or viruses, or otherpotentially infectious agents.

In some exemplary embodiments, the acellular tissue matrix particles maybe sterilized 107 prior to or after mixing 108 with the flowablecarrier. In one exemplary embodiment, the sterilization 107 may compriseelectron-beam sterilization, as is known in the art. Alternativesterilization techniques may also be employed, as is known in the art.

The acellular tissue matrix particles and flowable carrier can be mixed108 in any suitable way. In one exemplary embodiment, the acellulartissue matrix particles and flowable carrier may be mixed in a largevolume batch under generally sterile conditions to form a tissue productcomposition in accordance with the present invention. The mixing 108 maycomprise, for example, stirring the acellular tissue matrix particlesand flowable carrier together to form a slurry. The parameters andtechnique of the mixing 108 may be altered according to the propertiesof the flowable carrier and the acellular tissue matrix particles, aswell as the general amounts of each in the tissue product composition,and can be readily derived by one skilled in the art from routineexperimentation.

In some exemplary embodiments, the hyaluronic acid based material iscross-linked for stabilization. The cross-linking may occur before,after, or during mixing 108 with the acellular tissue matrix particles.In some exemplary embodiments, the material is cross-linked after freezedrying. However, the material can also be cross-linked before or duringthe freeze-drying process. Cross-linking may be performed in a varietyof ways. In one exemplary embodiment, cross-linking occurs by contactingthe hyaluronic acis based material with one or more cross-linking agentssuch as glutaraldehyde, genepin, carbodiimides (e.g.,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)),diisocyantes, or 1,4-butanediol diglycidyl ether (or1,4-bis(2,3-epoxypropoxy)butane or 1,4-bisglycidyloxybutane, all ofwhich are commonly known as BDDE. In addition, cross-linking may beperformed by heating the material. For example, in some embodiments, thematerial may be heated to between 70° C. to 120° C., or between 80° C.and 110° C., or to about 100° C., or any values between the specifiedranges in a reduced pressure or vacuum. In addition, other cross-linkingprocesses, or combination of processes may be used to produce any of thedisclosed cross-linked products, including ultraviolet irradiation,gamma irradiation, and/or electron beam irradiation. In addition, avacuum is not needed but may reduce cross-linking time. Further, loweror higher temperatures could be used as long as melting of the matrixproteins does not occur and/or sufficient time is provided forcross-linking.

In various embodiments, the cross-linking process may be controlled toproduce a tissue product composition with desired mechanical,biological, and/or structural features. For example, cross-linking mayinfluence the overall strength of the tissue product composition, andthe process may be controlled to produce a desired strength. Inaddition, the amount of cross-linking may affect the ability of thetissue product composition to maintain a desired shape and structure(e.g., porosity) when implanted. Accordingly, the amount ofcross-linking may be selected to produce a stable three-dimensionalshape when implanted in a body, when contacted with an aqueousenvironment, and/or when compressed (e.g., by surrounding tissues ormaterials).

Excessive cross-linking may change the extracellular matrix materials.For example, excessive cross-linking may damage collagen or otherextracellular matrix proteins. The damaged proteins may not supporttissue regeneration when the tissue product compositions are injected ina patient. In addition, excessive cross-linking may cause the materialto be brittle or weak. Accordingly, the amount of cross-linking may becontrolled to produce a desired level of stability, while maintainingdesired biological, mechanical, and/or structural features.

In some exemplary embodiments, seed cells may be introduced into theflowable carrier and/or acellular tissue matrix particles before,during, or after mixing 108. The seed cells may, in some embodiments,comprise cells from a patient, i.e., autologous cells, for seeding intothe tissue product composition prior to application of the tissueproduct composition. In some exemplary embodiments, the seed cells maybe cultured with the flowable carrier and acellular tissue matrixparticles prior to application; in other exemplary embodiments, the seedcells may be applied to the tissue product composition followingapplication. The seed cells may include, but are not limited to,adipocytes, various stem cells, blood cells, etc., which may promoteadhesion, infiltration, and/or growth of other cells to the injectedtissue product composition. Similarly, various growth factors or othersubstances can be included in the tissue product composition toencourage growth of cells to the injected tissue product composition.

As described previously, the tissue product compositions should have theability to support cell ingrowth and tissue regeneration when implantedin or on a patient. In addition, the tissue product compositions mayhave the ability to act as a carrier for and support the growth ofcells, such as autologous cells from the patient. Accordingly, theprocesses described herein should not alter the extracellular matrixproteins (e.g., by damaging protein structure and/or removing importantglycosaminoglycans and/or growth factors). In some embodiments, theproducts will have normal collagen banding as evidenced by microscopyand described further herein.

In various embodiments, the tissue products are treated with a processthat retains either or both of the native hyaluronic acid andchondroitin sulfate. Accordingly, the tissue products may include eitheror both of hyaluronic acid and chondroitin sulfate. In addition, theprocess may be selected to maintain native growth factors. For example,the tissue products may be produced such that the tissue productscontains one or more growth factors selected from PECAM-1, HGF, VEGF,PDGF-BB, follistatin, IL-8, and FGF-basic.

A. Exemplary Tissue Product Compositions

Various hyaluronic acid based materials may be mixed with acellulartissue matrix particles to produce various tissue product compositionsdescribed in Table 1 below, in accordance with the present invention. Itshould be appreciated that the hyaluronic acid based materials describedherein are exemplary only, and other hyaluronic acid based materials maybe mixed with the acellular tissue matrix particles. Further, thecompositions given in Table 1 are exemplary only, and other formulationsof tissue product compositions may be formed in accordance with thepresent invention.

To formulate the tissue product compositions described in Table 1, fourdifferent types of hyaluronic acid based materials were used. All HATypes used to form Compositions 1-11 were initially in a solution havinga concentration of 20 mg HA/mL. HA Type 1 is a non-crosslinkedhyaluronic acid having a G′ value of 320 Pa; HA Type 2 and HA Type 3, incontrast, are hyaluronic acids that were cross-linked with an EDCcross-linking agent with different G′ and G″ values, as can be seen inFIG. 4, depending on the degree of cross-linking. HA Type 2 had a G′value of 160 Pa and HA Type 3 had a G′ value of between 500-550. HA Type4 is also cross-linked, but uses BDDE as the cross-linking agent. HAType 4 may have a G′ value of 350 Pa.

TABLE 1 ADM ADM:HA HA Type [HA] [ADM] Composition Slurry Ratio (20mg/mL) (mg/mL) (mg/mL) 1 ADMS 9:1 HA1 2 135 2 ADMS 9:1 HA2 2 135 3 ADMS7:3 HA3 6 105 4 ADMS 7:3 HA4 6 105 5 ADMS 4:6 HA3 12 60 6 ADMS 4:6 HA412 60 7 ADMS 1:9 HA3 18 15 8 ADMS 5:5 HA1 10 75 9 ADMS 5:5 HA2 10 75 10ADMS 5:5 HA2 10 75 11 ADMS 19:1  HA1 1 142.5

Turning now to Table 1, exemplary embodiments of tissue productcompositions formed in accordance with the present invention aredescribed. Compositions 1-11, representing various tissue productcompositions are illustrated in Table 1, but it should be appreciatedthat other tissue product compositions may be formed in accordance withthe present invention. For each Composition 1-11, the acellular tissuematrix particles originated from porcine acellular dermal matrix (ADM)and, when combined with the flowable carrier, produced acellular dermalmatrix slurries (ADMS), which may also be referred to as “flowable ADM.”Prior to mixing with the flowable carrier, which was provided in aconcentration of 20 mg HA/mL, the acellular tissue matrix particles werein a concentration of 150 mg/mL. As should be appreciated from Table 1,a ratio of ADM:HA can be adjusted to produce slurries with varying flowproperties, as will be described further herein. It should be understoodthat the ratios described herein can be either by volume or by mass; inthe exemplary embodiments shown in Table 1, the ratio is given as volumeADM:volume HA. In some exemplary embodiments, the ratio of ADM:HA canvary from between 1:9 and 19:1. As exemplified by Compositions 1 and 2,the ratio of ADM:HA can be 9:1; as exemplified by Compositions 3 and 4,the ratio of ADM:HA can be 7:3; as exemplified by Compositions 5 and 6,the ratio of ADM:HA can be 4:6; as exemplified by Composition 7, theratio of ADM:HA can be 1:9; as exemplified by Compositions 8, 9, and 10,the ratio of ADM:HA can be 5:5; and as exemplified by Composition 11,the ratio of ADM:HA can be 19:1. It should be appreciated that thepreviously described ratios are exemplary only, and other exemplaryembodiments of tissue product compositions may have other ratios ofADM:HA, including values between the exemplary ratios.

According to certain aspects of this disclosure, a tissue productcomposition with a desired tissue matrix particle solid content may beused. For example, a material that is 2% to 20% solid content, such as10% to 15% solid content, may be desired depending on what type ofhyaluronic acid based material is mixed with the tissue matrixparticles. In some exemplary embodiments, the tissue product compositionhas 15% solid content, corresponding to 150 mg/mL, of acellular tissuematrix particles.

Referring again to FIG. 1, the method 100 of forming tissue productcompositions may further include sorting 105 the tissue matrix particlesby particle size. In some exemplary embodiments, the sorting 105 mayinclude straining the tissue matrix particles through one or moresieves. While the sorting 105 is shown as occurring afterde-cellularization 103 and prior to sterilization 107, it should beappreciated that the sorting 105 may occur before de-cellularization 103and/or after sterilization 107, if desired. Further, the tissue matrixparticles may be sorted in ways other than straining.

In one exemplary embodiment, the tissue matrix particles may be strainedthrough a first sieve defining a first sieve diameter and a second sievedefining a second sieve diameter that is less than the first sievediameter. The first sieve allows tissue matrix particles with particlesizes less than the first sieve diameter to pass through, while thesecond sieve allows tissue matrix particles with particle sizes lessthan the second sieve diameter to pass through. In this sense, a firstmixture of sorted tissue matrix particles left in the first sieve candefine a first average particle size greater than the first sievediameter, and a second mixture of sorted tissue matrix particles left inthe second sieve can define a second average particle size less than thefirst sieve diameter but greater than the second sieve diameter.

The first mixture of sorted tissue matrix particles and the secondmixture of sorted tissue matrix particles may be combined 106 to producea tissue matrix particle mixture with a desired size distribution. Itshould be appreciated that more than two sieves each defining arespective sieve diameter can be used to sort 105 the tissue matrixparticles by particle size. In one exemplary embodiment, tissue matrixparticle sizes may range from 50 microns to 3,500 microns. For example,the tissue matrix particles may be sieved to retrieve particles with thefollowing dimensions: Extra fine particles (e.g., 50-100 microns); Fineparticles (e.g., 100-400 microns); Medium particles (e.g., 0.4 mm to 0.6mm); Large particles (e.g., 0.8 mm to 1 mm); and Larger particles(e.g., >1 mm). In some aspects of the present disclosure, particle sizesin this range may not invoke a varied biological response. In otherwords, for example, there may be no difference in biological responseswith particle sizes ranging from 50 microns to 3,500 microns. Differentapplications that may require a specific size of an injection needle mayselect a specific size of particle(s) without the need to consider ifthe biological responses will be different.

Referring now to FIG. 2, exemplary tissue matrix particle sizedistributions are illustrated. As can be seen, the graph illustrates afirst particle size plot 201, a second particle size plot 202, and athird particle size plot 203 of various tissue product compositionsformed in the accordance with the present invention. The first particlesize plot 201 shows a first tissue product composition having a majorityof particle sizes ranging from 200 to 2000 μm; the second particle sizeplot 202 shows a second issue product composition having a majority ofparticle sizes ranging from 200 to 2000 μm; and the third particle sizeplot 203 shows a third tissue product composition having a majority ofparticle sizes ranging from 500 to 2500 μm, as shown. Controlling thetissue matrix particle size distribution can allow adjustment of flowand/or spread properties of the tissue product composition.

B. Properties of Exemplary Tissue Product Compositions

To determine how the properties of tissue product compositions formed inaccordance with the present invention compared to pure acellular tissuematrix sheets and/or particles, various tests were conducted to measureproperties of the tissue product compositions. One such test that wasperformed was differential scanning calorimetry (DSC) to determine theonset temperature of various exemplary embodiments of tissue productcompositions including a flowable carrier comprising a hyaluronic acidbased material and acellular tissue matrix particles. From DSC analysis,it was found that the tested tissue product compositions with theflowable carrier and acellular tissue matrix particles can have an onsettemperature in a range between 55° C. and 60° C., which is similar tothe onset temperature (56° C.-58° C.) of sheet acellular dermal matrix.

Referring now to FIGS. 3 and 4, rheology analyses of various tissueproduct compositions are illustrated. FIG. 3 illustrates an amplitudesweep report of acellular dermal matrix in a flowable (slurry) formhaving 15% solid content, corresponding to a concentration of 150 mg/mL.The frequency sweep used was 0.3% strain to determine an elastic(“storage”) modulus (G′) plot 301, a viscous (“loss”) modulus (G″) plot302, and a phase angle (δ) plot 303 as functions of complex sheer strainpercentages. As is known in rheology, G′ generally indicates the abilityof a material to recover its shape after deformation, i.e., elasticity,while G″ generally indicates the viscosity of a material.

Referring now to FIG. 4, G′ and G′ values of various tissue productcompositions and flowable carriers are illustrated. The G′ and G″ valuesin FIG. 4 were determined using amplitude sweep at a frequency of 5 Hz;the G′ value is shown on the left for each material, while the G″ valueis shown on the right for each material. As can be seen, the G′ and G″values for pure tissue matrix are higher than respective values of anyone of Compositions 1-7 including tissue matrix particles mixed within aflowable carrier comprising a hyaluronic acid based material or HA Types1-4. Particularly, the viscosity of Compositions 1-7 are significantlylower than the viscosity of the pure tissue matrix particles. The G″value for Compositions 1-7, as shown, are less than 600 Pa, with some ofthe compositions having G″ values less than 500 Pa.

FIG. 5 illustrates a graph 500 showing compressive load as a function ofcompressive extension for various tissue product compositions injectedusing a syringe with an 18 gauge needle. As can be seen, the load vs.extension plots 501, 502, 503 for various injections of pure acellulartissue matrix particles all have large degrees of compressive loadfluctuation during the injection and peak compressive loads above 10Nduring the injection. In contrast, load vs. extension plot 504 for aninjection of a tissue product composition comprising acellular tissuematrix particles mixed with HA Type 1 in an ADM:HA ratio of 9:1, i.e.,the tissue product composition is 90% acellular tissue matrix particlesand 10% flowable carrier, has a relatively constant and low compressiveload during the injection. It should therefore be appreciated thatmixing the acellular tissue matrix particles with the flowable carriercan both reduce the peak compressive loads on the tissue matrixparticles during injection and make the injection “smoother.”

Referring to FIG. 6, a graph 600 illustrates maximum compressive loadsfor various tissue product compositions injected through an 18 gaugeneedle and, where feasible, through a 21 gauge needle. Each trial placed1 mL of tissue product composition in a reservoir for injection throughan 18 gauge or 21 gauge needle by depressing a plunger, with the plungerbeing depressed a total of 40 mm at a speed of 1 mm/sec. As is known, a21 gauge needle has a significantly reduced inner diameter compared toan 18 gauge needle. The maximum compressive load for an 18 gauge needleis shown on the left for each material, while the maximum compressiveload for a 21 gauge needle is shown on the right for each material; amaximum compressive load for injection of pure acellular tissue matrixthrough a 21 gauge needle is not shown because the particles aregenerally too large to pass through a 21 gauge needle in pure form. Ascan be seen in FIG. 6, the maximum compressive load for injecting pureacellular tissue matrix particles through an 18 gauge needle wassignificantly higher than the maximum compressive load for injecting anyone of Compositions 1-7, formed in accordance with the presentinvention, through an 18 gauge needle. A maximum compressive load forinjecting pure acellular tissue matrix particles through a 21 gaugeneedle is not illustrated. It was found that injecting Compositions 1,2, 4, and 6 comprising acellular tissue matrix particles and a flowablecarrier through a 21 gauge needle produced a lower maximum compressiveload than injecting pure acellular tissue matrix particles through asignificantly larger 18 gauge needle. Injection of Compositions 3, 5,and 7 was also possible through a 21 gauge needle, although therespective maximum compressive loads were found to be higher than forCompositions 1, 2, 4, and 6. It should therefore be appreciated thatmixing acellular tissue matrix particles within a flowable carriercomprising a hyaluronic acid based material can produce a tissue productcomposition suitable for injection through a 21 gauge needle.

C. The Tissue Product Compositions have Intact Collagen Structure

Referring now to FIGS. 7-15, various microscope images are shown toillustrate the general structure and distribution of various tissueproduct compositions.

Referring specifically to FIG. 7, trichrome staining of a tissue productcomposition including acellular tissue matrix particles mixed within aflowable carrier comprising a hyaluronic acid based material is shown.As can be seen in FIG. 7, there is no appreciable damage of the collagenfibers and the collagen has normal banding patterns.

Referring specifically to FIG. 8, a microscope image of Haemotoxylin andEosin (H&E) staining of pure acellular tissue matrix particles is shown.As can be seen, the collagen fibers are normal.

Referring specifically to FIG. 9, a microscope image of H&E staining ofa tissue product composition including a 5:5 ratio of acellular tissuematrix particles to carrier HA4 is shown. As can be seen, the collagenfibers are normal and the HA4 molecules tend to congregate about theperipheries of the particles.

Referring specifically to FIG. 10, a microscope image of H&E staining ofa tissue product composition including a 9:1 ratio of acellular tissuematrix particles to carrier HA2 is shown. As can be seen, the collagenfibers are normal and the HA2 molecules tend to be interspersed betweenthe particles.

Referring specifically to FIG. 11, a microscope image of H&E staining ofa tissue product composition including a 1:9 ratio of acellular tissuematrix particles to carrier HA3 is shown. As can be seen, the collagenfibers are normal and the HA3 molecules surround the particles.

Referring specifically to FIG. 12, a microscope image of Alcian Bluestaining of pure acellular tissue matrix particles is shown. As can beseen, the collagen fibers are normal.

Referring specifically to FIG. 13, a microscope image of Alcian Bluestaining of a tissue product composition including a 5:5 ratio ofacellular tissue matrix particles to carrier HA4 is shown. As can beseen, the collagen fibers are normal and the HA4 molecules tend tocongregate about the peripheries of the particles.

Referring specifically to FIG. 14, a microscope image of Alcian Bluestaining of a tissue product composition including a 9:1 ratio ofacellular tissue matrix particles to carrier HA2 is shown. As can beseen, the collagen fibers are normal and the HA2 molecules tend to beinterspersed between the particles.

Referring specifically to FIG. 15, a microscope image of Alcian Bluestaining of a tissue product composition including a 1:9 ratio ofacellular tissue matrix particles to carrier HA3 is shown. As can beseen, the collagen fibers are normal and the HA3 molecules surround theparticles.

It should therefore be appreciated that tissue product compositionsincluding a flowable carrier comprising a hyaluronic acid based materialand acellular tissue matrix particles mixed within the carrier can havesuitable structure for supporting tissue growth.

D. In Vivo Implantation of the Tissue Product Compositions UsingHyaluronic Acid (HA) as a Carrier

The disclosed tissue product compositions using hyaluronic acid (HA) asa flowable carrier have certain advantages when used as in vivo implantsin a host dermal tissue.

Referring specifically to FIG. 16, acellular tissue matrix particles(15% solid content) were mixed with a 20 mg/mL (2%) non-crosslinked HAcarrier in a 19:1 volume ratio. The final solid content of the acellulartissue matrix particles was 14.25%, and the final concentration of theHA carrier was 0.1%. For tissue implantation, 120 μL of the mixture wasinjected into the subdermal space between the dermis and muscle layersof a rat's dorsal area. Five rats were tested with 4 testing arms each.An explantation was performed 4 weeks post implantation, and the explantwas placed in 15 mL of 10% formalin for histology processing followed byan H&E staining. Images were taken under 20× magnification. As shown inFIG. 16, the implanted tissue product with HA as a carrier integratedwell into the host dermal tissue 4 weeks post implantation. Notablecellular infiltration with fibroblasts and vascularization occurred withlittle or no inflammation.

Referring to FIG. 17, the biological responses in tissue implants withor without HA as a carrier were tested. Acellular tissue matrixparticles with a 15% solid content was used. When using HA as a carrier,the acellular tissue matrix was mixed with a 20 mg/mL (2%)non-crosslinked HA carrier in a 9:1 volume ratio. The final solidcontent of the acellular tissue matrix particles was 13.5% and the finalconcentration of HA was 0.2%. For tissue implantation, 500 μL ofacellular tissue matrix particles alone, or the mixture of acellulartissue matrix particles with HA as a carrier was injected subcutaneouslyinto a rat's dorsal area. Five rats were tested with 4 testing armseach. An explantation was performed 4 weeks or 12 weeks postimplantation, and the explants were placed in 15 mL of 10% formalin forhistology processing followed by an H&E staining. Images were takenunder 10× magnification. Biological responses such as cell infiltration,vascularization, and minimal inflammation were examined. As shown inFIGS. 17A-D, similar biological responses were observed in tissueexplants with the acellular tissue matrix alone, and the acellulartissue matrix with HA as a carrier. These results demonstrate that usingHA as a carrier does not adversely impact the host's biologicalresponses to the tissue implants, and the results last for at least 12weeks post implantation.

Referring specifically to FIGS. 18A and 18B, increase of dermalthickness in implanted area was tested. Acellular tissue matrixparticles (15% solid content) were mixed with a 20 mg/mL (2%)non-crosslinked HA carrier in a 19:1 volume ratio. The final solidcontent of the acellular tissue matrix particles was 14.25% and thefinal concentration of the HA carrier was 0.1%. For tissue implantation,120 μL of the mixture was injected into the subdermal space between thedermis and muscle layers of a rat's dorsal area. Five rats were testedwith 4 testing arms each. An explantation was performed 4 weeks or 12weeks post implantation, and the explants were placed in 15 mL of 10%formalin for histology processing followed by an H&E staining. As shownin FIG. 18, the implantation of the acellular tissue matrix with HA as acarrier significantly increased the thickness of the dermal tissue, andthe increase in dermal thickness lasts as long as 12 weeks postimplantation (FIG. 18B).

E. Volume Retention of Implanted Tissue Product Compositions

Referring specifically to FIG. 19, volume retention of the implantedacellular tissue matrix was tested. For tissue implantation, 500 μL ofacellular tissue matrix particles (15% solid content) were injectedsubcutaneously into a rat's dorsal area. An explantation was performed 4weeks, 12 weeks, or 24 weeks post implantation. For the 4-weekexplantation, 10 rats were tested with 4 testing arms each. For the12-week and 24-week explantations, 5 rats were tested for each groupwith 4 testing arms in each rat. Explants were harvested and weighted.As shown in FIG. 19, the implanted acellular tissue matrix retained atleast 90% of its original volume (dotted line) well for at least 24weeks post implantation.

Referring specifically to FIG. 20, the acellular tissue matrix particleswas treated with different concentrations of1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)(0.00625%, 0.0125%, 0.025%, and 0.05%) for 18 hours to form cross-linkedtissue products. The cross-linked products were subjected to a finalsterilization and washed with PBS for 3 times, 30 min each. Tube weightand wet tissue weight were recorded before the tissues were treated with1250 U/mL collagenase for 4 h, 8 h, and 16 h, respectively. Tripletsamples were tested for each condition. After incubation, the remainingtissues were dried overnight and the weight of the dried tissues in thetube were recorded. The ratios of weight for the dried tissues over thewet tissues before enzymatic treatment were calculated for each timepoint. As shown in FIG. 20, the cross-linked acellular tissue matrix hadsignificantly increased collagenase resistance compared tonon-crosslinked acellular tissue matrix. Higher concentrations of thecross-linker resulted in higher collagenase resistance for the sameperiod of incubation.

Referring specifically to FIGS. 21 and 22, cross-linked acellular tissuematrices were tested for in vivo implantation. 500 μL of acellulartissue matrix particles (15% solid content) were cross-linked with0.0125% (modified L) or 0.05% (modified H) EDC, respectively, andinjected subcutaneously into a rat's dorsal area. Five rats were testedfor each condition, with 4 testing arms in each rat. An explantation wasperformed 4 weeks or 24 weeks post implantation. Explants were harvestedand weighted. The harvested explants were stained by H&E staining andrepresentative images were taken under 20× magnification. As shown inFIG. 21, the volumes of the implanted tissue matrices were maintainedwell for at least 24 weeks, and higher level of cross-linking resultedin better retention of volume. As shown in FIG. 22, similar biologicalresponses were observed in explants with unmodified, modified L, andmodified H tissue matrices.

F. Exemplary Uses of the Tissue Product Composition

In one exemplary embodiment provided in accordance with the presentinvention, a tissue product composition including a flowable carriercomprising a hyaluronic acid based material and acellular tissue matrixparticles mixed within the carrier may be loaded into a syringe to forman injection device. In some exemplary embodiments, the tissue productcomposition may be any of the previously described tissue productcompositions. The syringe generally includes a reservoir defining avolume and a needle fluidly coupled to the reservoir. The tissue productcomposition is held in the reservoir, and in some exemplary embodimentsmay entirely fill the volume of the reservoir, which may be any desiredvolume, such as between 0.5 mL and 5 mL. For certain procedures, areservoir with a considerably larger volume, such as 200 mL, may beused. The inner diameter of the needle may be selected, as desired,according to the injection site and procedure being performed. In someexemplary embodiments, the needle may be an 18 gauge needle, a 19 gaugeneedle, a 20 gauge needle, a 21 gauge needle, or a higher gauge (lowerinner diameter) needle. The injection device may be pre-filled with thetissue product composition and kept in storage prior to being injected.

Alternatively, the injection device may be formed during the procedureby a user, such as a physician, by loading the tissue productcomposition into an empty syringe. Further, the disclosed compositionscan be provided as a kit, wherein the tissue matrix particles are heldin a container separate from the carrier or HA component, and a user maymix the component prior to use. The kit can include two or moresyringes, two or more vials, a vial/syringe combination, or amulti-compartment system that allows easy mixing at the time of use. Itshould be appreciated that while the injection device is described as asyringe, the injection device may take other forms suitable forinjecting the tissue product composition into a patient.

In one exemplary embodiment provided in accordance with the presentinvention, a method of treating a patient is provided. The methodincludes injecting a tissue product composition into a body of thepatient, the tissue product composition including a flowable carriercomprising a hyaluronic acid based material and acellular tissue matrixparticles mixed within the carrier. In some exemplary embodiments, thetissue product composition may be any of the previously described tissueproduct compositions. For cosmetic treatments, the tissue productcomposition may be an injectable composition that is injected into oneor more layers the skin of the patient to, for example, reduce theappearance of wrinkles or other lines in the skin. Further, in otherexemplary embodiments, the tissue product composition may be formed as apaste or putty that can be applied to other anatomical locations in thepatient to restore and/or repair large volumes of lost and/or damagedtissue. It should therefore be appreciated that the tissue productcompositions formed in accordance with the present invention can betailored to many different applications and applied in a variety ofdifferent ways.

While principles of the present disclosure are described herein withreference to illustrative embodiments for particular applications, itshould be understood that the disclosure is not limited thereto. Thosehaving ordinary skill in the art and access to the teachings providedherein will recognize additional modifications, applications,embodiments, and substitution of equivalents all fall within the scopeof the embodiments described herein. Accordingly, the invention is notto be considered as limited by the foregoing description.

1. A tissue product composition, comprising: a flowable carriercomprising a hyaluronic acid based material; and a plurality ofacellular tissue matrix particles mixed within the carrier.
 2. Thecomposition of claim 1, wherein the hyaluronic acid based material is across-linked hyaluronic acid.
 3. The composition of claim 1, wherein thehyaluronic acid based material is a non-cross-linked hyaluronic acid. 4.The composition of claim 1, wherein the carrier has a concentration ofabout 20 mg/mL hyaluronic acid prior to mixing.
 5. The composition ofclaim 1, wherein the tissue matrix particles originate from a dermalmatrix.
 6. The composition of claim 5, wherein the tissue matrixparticles originate from a dermal matrix and an adipose matrix.
 7. Thecomposition of claim 1, wherein the composition defines a ratio of thetissue matrix particles to the carrier, the ratio being in the range ofbetween 1 and 9:between 19 and
 1. 8. The composition of claim 7, whereinthe ratio is 19:1.
 9. The composition of claim 7, wherein the ratio is9:1.
 10. The composition of claim 7, wherein the ratio is 7:3.
 11. Thecomposition of claim 7, wherein the ratio is 5:5.
 12. The composition ofclaim 7, wherein the ratio is 4:6.
 13. The composition of claim 7,wherein the ratio is 1:9.
 14. The composition of claim 1, wherein thecarrier and tissue matrix particles are mixed to form a slurry withabout 1.5% to about 14.25% solid content.
 15. The composition of claim14, wherein the slurry has a G″ value at 5 Hz that is less than 600 Pa.16. The composition of claim 15, wherein the G″ value at 5 Hz is lessthan 500 Pa.
 17. The composition of claim 1, further comprising aplurality of seed cells intermixed with the carrier and tissue matrixparticles.
 18. An injection device, comprising: a syringe including areservoir defining a volume and a needle fluidly coupled to thereservoir; and a tissue product composition held in the reservoir, thetissue product composition comprising: a flowable carrier comprising ahyaluronic acid based material; and a plurality of acellular tissuematrix particles mixed within the carrier.
 19. The injection device ofclaim 18, wherein the hyaluronic acid based material is a cross-linkedhyaluronic acid.
 20. The injection device of claim 18, wherein thehyaluronic acid based material is a non-cross-linked hyaluronic acid.21. The injection device of claim 18, wherein the carrier has aconcentration of about 20 mg/mL prior to mixing. 22.-44. (canceled)